Follicle stimulating hormone-glycosylation analogs

ABSTRACT

The invention provides recombinant native and mutein forms of human follicle stimulating hormone beta subunit (FSH beta) with characteristic glycosylation patterns which are influential in the metabolic activity of the protein. The invention also provides recombinant mutant forms of the human alpha subunit common to FSH, LH, CG, and TSH, to obtain hormones which also have unique glycosylation patterns. Also provided are recombinant materials to produce these subunits separately or together to obtain complete heterodimeric hormones of regulated glycosylation pattern.

TECHNICAL FIELD

[0001] The invention relates to the production of follicle stimulatinghormone (FSH) with altered glycosylation patterns and activities. Inparticular, it concerns production of recombinant FSH under conditionswhich regulate the glycosylation pattern of the protein.

BACKGROUND ART

[0002] Human FSH is used therapeutically to regulate various aspects ofmetabolism pertinent to reproduction in the human female. For example,FSH partially purified from urine is used clinically to stimulatefollicular maturation in anovulatory women with anovulatory syndrome orluteal phase deficiency. It is also used in combination with luteinizinghormone (LH) to stimulate the development of ovarian follicles for invitro fertilization. The role of FSH in the reproductive cycle issufficiently well-known to permit this sort of therapeutic use, butdifficulties have been encountered due, in part, to the heterogeneity ofthe preparation from native sources. This heterogeneity is due tovariations in glycosylation pattern.

[0003] FSH is one member of a family of heterodimeric human glycoproteinhormones which have a common alpha subunit, but differ in theirhormone-specific beta subunits. The family includes, besides FSH,luteinizing hormone (LH), thyrotropin or thyroid stimulating hormone(TSH), and human chorionic gonadotropin (CG). In all cases, the alphasubunit is a 92 amino acid glycoprotein with two canonical glycosylationsites at the asparagines located at positions 52 and 78. The betasubunits are also glycoproteins; in addition to the N-linkedglycosylation exhibited by the beta chains of all four hormones, humanCG contains four mucin-like O-linked oligosaccharides attached to acarboxy-terminal extension unique to this hormone. The relevance of theO-linked glycosylation is not, apparently, related to the secretion andassembly of the hormone (Matzuk, M. M. et al. Proc Natl Acad Sci USA(1987) 84:6354-6358).

[0004] Genomic and cDNA clones have been prepared corresponding to thehuman alpha chain (Boothby, M. et al. J Biol Chem (1981) 256:5121-5127;Fiddes, J. C. et al. J Mol App Genet (1981) 1:3-18). The cDNA andgenomic sequences of the beta subunits of the remaining three members ofthe family have also been prepared: for CG, as disclosed by Fiddes, J.C. et al. Nature (1980) 286:684-687 and by Policastro, P. et al. J BiolChem (1983) 258:11492-11499; for luteinizing hormone by Boorstein, W. R.et al. Nature (1982) 300:419-422; and for TSH by Hayashizaki, Y. et al.FEBS Lett (1985) 188:394-400 and by Whitfield, G. K. et al. in“Frontiers in Thyroidology”, (1986) Medeiros-Nato, G. et al. (eds) pages173-176, Plenum Press, N. Y. These DNA segments have been expressedrecombinantly, and biologically active material has been produced.

[0005] Although genomic clones and isolates for human FSH-beta hve beenprepared (Watkins, P. C. et al. DNA (1987) 6:205-212; Jameson, J. L. etal., Mol Endocrinol (1988) 2:806-815; Jameson, J. L. et al. J ClinEndocrinol Metab (1986) 64:319-327; Glaser, T. et al. Nature (1986)321:882-887), human FSH beta has not been engineered to permitrecombinant production of the hormone. (The bovine beta FSH gene hasalso been obtained as disclosed in Maurer, R. A. et al. DNA (1986)5:363-369; Kim, K. E. et al. DNA (1988) 7:227-333.) As disclosed in theinvention herein, recombinant production of this FSH hormone permitsregulation of the glycosylation pattern and thereby greaterpredictability in the formulation of therapeutically useful material.

[0006] While it is now understood that the glycosylation pattern of aparticular protein may have considerable relevance to its biologicalactivity, the importance of this pattern has largely been overlooked incharacterization of glycoproteins. Emphasis has been placed on the aminoacid sequence as if this were the sole component of the glycoprotein.The reasons for this myopia are largely historic, but this almostexclusive focus on the peptide aspect is clearly in error. For example,it is well known in the case of human CG that desialylation causes thehormone to be cleared rapidly via the liver (Morell, A. G. et al. J BiolChem (1971) 246:1461-1467). It is also known that removal ofcarbohydrate internal to the sialic acid residues or completedeglycosylation converts human CG into an antagonist which binds moretightly to receptor but shows decreased biological activity in vitro(Channing, C.P. et al. Endocrinol (1978) 103:341-348; Kalyan, N. J. etal. J Biol Chem (1983) 258:67-74; Keutmann, H. T. et al. Biochemistry(1983) 3067-3072; Moyle, W. R. et al. J Biol Chem (1975) 250:9163-9169).Other glycoproteins, such as, for example, tissue plasminogen activator,are also known to be altered in their degree of activity when theglycosylation pattern is changed. Therefore, it appears that in order toregulate the therapeutic function of the glycoprotein hormones, it maybe necessary to control both the level and nature of glycosylation.

[0007] Disclosure of the Invention

[0008] The invention provides recombinantly produced human FSH whichoffers the opportunity for control of gly-cosylation pattern both on thealpha and beta portions of the heterodimer. Such glycosylation controlcan be obtained through either the prudent selection of the recombinanteucaryotic host, including mutant eucaryotic hosts, or throughalteration of glycosylation sites through, for example, site directedmutagenesis at the appropriate amino acid residues. In any event, therecombinant production of this hormone obviates the complex mixture ofglycosylation patterns obtained when the hormone is isolated from nativesources.

[0009] In one aspect, the invention is directed to expression systemscapable, when transformed into a suitable host, of expressing the geneencoding the FSH beta subunit. In additional aspects, the invention isdirected to recombinant hosts which have been transformed or transfectedwith this expression system, either singly, or in combination with anexpression system capable of producing the alpha subunit. In otheraspects, the invention is directed to the FSH beta monomers and FSHheterodimers of defined glycosylation pattern produced by therecombinant host cells.

[0010] In another aspect, the invention is directed to specific mutantsof FSH or other hormones of this family with altered glycosylationpatterns at the two glycosylation sites in the alpha subunit, or toalpha subunit variants containing alterations at the carboxy terminuswhich affect activity and to glycosylation or other variants of the FSHbeta subunit. Thus, in another aspect, the invention is directed toexpression systems for the alpha subunit which lack glycosylation sitesat the asparagine at position 52 or position 78 or both, for the FSHbeta subunit and its variants, and to recombinant host cells transfectedwith these expression systems. The cells may be transfected with asubunit expression system singly or in combination with an expressionsystem for a suitable alpha or beta subunit. The invention is directedalso to the mutant glycoproteins with altered glycosylation or activitypatterns produced by these cells.

[0011] In other aspects, the invention is directed to pharmaceuticalcompositions containing the variants set forth above, and to methods toregulate reproductive metabolism in subjects by administration of theseglycoproteins or their pharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows a restriction enzyme map of the human FSH beta gene.

[0013]FIG. 2 shows the nucleotide sequence of the human FSH beta gene.

[0014]FIG. 3 shows expression vectors for production of human beta FSH.

[0015]FIG. 4 shows 35-S cysteine labeled FSH or FSH betaimmunoprecipitated from cell lysates and media.

[0016]FIG. 5 shows the bioassay of recombinant human FSH.

[0017]FIG. 6 shows chromatofocusing of recombinant and pituitary humanFSH.

[0018]FIG. 7 shows the construction of expression vectors for the humanalpha subunit.

MODES OF CARRYING OUT THE INVENTION

[0019] Definitions

[0020] As used herein, human alpha subunit, and human FSH, LH, TSH, andCG beta subunits as well as the heterodimeric forms have in generaltheir conventional definitions and refer to the proteins having theamino acid sequences known in the art per se, or allelic variantsthereof, deliberately constructed muteins thereof maintaining theactivity of the native protein regardless of the glycosylation patternexhibited, or mutant forms thereof having at least 90% homology with thenative forms. “Native” forms of these peptides are those which have theamino acid sequences isolated from human tissue, and have these knownsequences per se, or their allelic variants. “Mutein” forms of theseproteins are those which have deliberate alterations in amino acidsequence produced by, for example, site-specific mutagenesis or by otherrecombinant manipulations, or which are prepared synthetically. Thesealterations result in amino acid sequences wherein the biologicalactivity of the subunit is retained and/or wherein the subunit has atleast 90% homology with the native form. A particularly preferred muteinof FSH beta, for example, is that wherein the amino acid carboxyterminal peptide (CTP) of hCG is fused to the carboxy terminus of FSHbeta. A preferred mutein of the alpha subunit for use in antagonists ofthe various heterodimers has alterations in the amino acids of positions88-92.

[0021] Although it is recognized that glycosylation pattern has aprofound influence on activity both qualitatively and quantitatively,for convenience the terms FSH, LH, TSH, and CG beta subunits refers tothe amino acid sequence characteristic of the peptides, as does “alphasubunit”. When only the beta chain is referred to, the terms will be,for example, FSH beta; when the heterodimer is referred to, the simpleterm “FSH” will be used. It will be clear from the context in whatmanner the glycosylation pattern is affected by, for example,recombinant expression host or alteration in the glycosylation sites.Forms of the glycoprotein with specified glycosylation patterns will beso noted.

[0022] A “transfected” recombinant host cell, i.e., a cell “transfected”with the recombinant expression systems of the invention, refers to ahost cell which has been altered to contain this expression system byany convenient manner of introducing it, including transfection, viralinfection, and so forth. “Transfected” refers to cells containing thisexpression system whether the system is integrated into the chromosomeor is extrachromosomal. The “transfected” cells may either be stablewith respect to inclusion of the expression system or not. In short,“transfected” recombinant host cells with the expression system of theinvention refers to cells which include this expression system as aresult of their manipulation to include it, when they natively do not,regardless of the manner of effecting this incorporation. “Expressionsystem” refers to a DNA sequence which includes a coding sequence to beexpressed and those accompanying control DNA sequences necessary toeffect the expression of the coding sequence. Typically, these controlsinclude a promoter, termination regulating sequences, and, in somecases, an operator or other mechanism to regulate expression. Thecontrol sequences are those which are designed to be functional in aparticular target recombinant host cell and therefore the host cell mustbe chosen so as to be compatible with the control sequences in theconstructed expression system.

[0023] As used herein “cells”, “cell cultures”, and “cell lines” areused interchangeably without particular attention to nuances of meaning.Where the distinction between them is important, it will be clear fromthe context. Where any can be meant, all are intended to be included.

[0024] Isolation of the Gene Encoding FSH Beta

[0025] An important aspect of the present invention is the provision ofan FSH beta-encoding DNA which is readily manipulated for insertion intoexpression systems. The gene, suitable for inclusion in expressionsystems intended for host cells capable of processing introns, wasprepared as follows:

[0026] Genomic DNA from JAr choriocarcinoma cells (a human placentaldonor) was partially digested with MboI and cloned into the BamHI siteof lambda MG3, a vector described Helms, C., et al. DNA (1985) 4:39-49;this vector is a derivative of lambda L47 which is described by Loenen,W. A. M., et al. Gene (1980) 10:249-259. The size of the inserts wastypically 15-20 kb. Approximately 5×10⁵ plaques were obtained andscreened according to the method of Benton, W. D., et al. Science (1977)196:180-182 using the 41 mer encoding amino acids 94-107 of exonIII ofhuman FSH beta as described by Watkins, P. C., et al. DNA (1987)6:205-212. This 41 mer has the sequence:

[0027] TGTACTGTGCGGGCCTGGGGCGGAGCTACTGCTCCTTTGG.

[0028] Two positive clones were isolated by repeated plaque purificationand shown by restriction analysis to be identical; furthermore, the PstIcleavage patterns were consistent with those obtained by Glaser, T. etal. Nature (1986) 321:882-887 (supra). Restriction fragments weresubcloned into pUC18 for further restriction analysis and into M13 forsequencing by the dideoxy chain termination method of Sanger, F., ProcNatl Acad Sci USA (1977) 74:5463-5467. A 3.7 kb HindIII/BamHI fragmentcontained in the 16.5 kb insert of these clones contains the hFSH betacoding sequence.

[0029] The clones were designated lambdai and lambdaR, and haveidentical restriction maps and are approximately 16.5 kb in length. Therestriction map of the full length clones are shown in FIG. 1, alongwith a restriction map of the 3.7 kb human FSH beta coding sequence.

[0030] The results of sequencing the human FSH beta gene are shown inFIG. 2. As shown in FIG. 2, the coding sequence is divided into threeexons. ExonI contains a 5′ untranslated tract previously reported toencode two transcripts of either 33 or 63 bp (Jameson, J. L. et al. MolEndocrinol (1988) 2:806-815). ExonII encodes an 18 amino acid signalpeptide and amino acids 1-35 of the mature protein. ExonIII encodesamino acids 36-111 and about 1.1 kb of 3′ untranslated sequence. ExonsIand II are separated by an intron of about 800 bp, and ExonsII and IIIby an intron of about 1.4 kb.

[0031] The nucleotide sequence obtained is similar to that reported byWatkins, T. C. et al. DNA (1987) 6:205-212 and Jameson, J. L. et al.(supra), except that tyrosine 58 is encoded by TAC rather than TAT andthere are differences from Watkins in the 3′ and 5′ untranslatedregions. A putative transcriptional start site 32 bp downstream from theTATA element is assigned by analogy to the bovine gene reported by Kim,K. E., et al., DNA (1988) 7:227-333. The sequence in FIG. 2 shows asingle polyadenylation signal (AATAAA) overlapping the termination codonand recent evidence from the bovine gene (supra) and human clones(Jameson, J. L. et al., (supra)) indicates the presence of anapproximately 1.1 kb 3′ untranslated tract which may contain alternatepolyadenylation signals.

[0032] The amino acid sequence shown in FIG. 2 is identical to thatreported by that of Watkins (supra) but differs from that reportedearlier by protein sequencing of purified human FSH beta. The carboxyterminal sequence Tyr-Pro-Thr-Ala-Leu-Ser-Tyr reported by Saxena, D. B.,J Biol Chem (1976) 251:993-1005 is found neither in the sequence shownin FIG. 2 nor in the protein based sequence reported by Shome, B., etal., J Clin Endocrinol Metab (1974) 39:203-205. A more recentdetermination of the amino acid sequence confirms the sequence deducedfrom the DNA (Stone, B. et al. J Prot Chem (1988) 7:325-339.

[0033] Construction of Expression Vectors for Native Human Alpha Subunitand its Muteins

[0034] It is understood in the art that N-linked glycosylation occurs atthe tripeptide site Asn-X-Thr/Ser, two of which sites occur in the humanalpha subunit, at Asn52 and Asn78. Site-directed mutagenesis wasperformed on a human alpha subunit fusion gene to alter these sites,wherein the fusion gene was constructed as follows:

[0035] The alpha subunit CDNA is obtained as described by Matzuk, M. M.et al. J Cell Biol (1988) 106:1049-1058 (supra) as a BamHI/XhoI framednucleotide sequence containing an XbaI site in the coding sequence. Agenomic fragment bounded by EcoRI and a XhoI site containing exons IIIand IV, with an XbaI site in exonIII was obtained from the humanchoriocarcinoma library. XbaI/XhoI digestion of both the genomicfragment and alpha subunit cDNA, followed by religation at the XbaI sitecreates the alpha subunit mini gene as a BamHI/XhoI fragment, containinga BglII site derived from the genomic fragment downstream of ExonIV. TheBamHI/BglII fragment digested from the mini gene is used as the alphasubunit-encoding insert in the construction of expression vectors; theBamHI/XhoI fragment itself is ligated into M13 UM20 for site-directedmutagenesis.

[0036] For alteration of Asn52 and Asn78, respectively, the 22-meroligomers GGTGACGTCCTTTTGCACCAAC and CTTAGTGGAGCGGGATATG respectivelywere used. This resulted in a substitution of aspartate residues forasparagine. Three mutants were constructed: αΔAsn-1 (position 52),αΔAsn-2 (position 78), and αΔAsn-1+2 (both positions). Correspondingchanges were made by substituting the codon for alanine in place of thatfor threonine at positions 54 and 80 using the 26 mers:GTGGACTCTGAGGCCACGTTCTTTTG and CAGTGGCACGCCGCATGGTTCTCCAC, respectivelyto obtain αΔThr1, αΔThr2 and αΔThr(1+2).

[0037] The wild type or mutant alpha subunits were then ligated into thehost vector pM² as 2.4 kb mini genes, using the BamHI/BglII fragmentsand were placed under control of the LTR promoter by insertion into theBamHI site downstream of the LTR. The construction of this expressionvector having the human alpha sequence under control of LTR is shown inFIG. 7. The resulting vector shown, pM²/CGα is then used as the sourceof the human alpha expression unit in pM²/α by excising this unit as anEcoRI/EcoRI fragment and ligating it into the EcoRI site of pM² (Matzuk,M. M. et al. Mol Endocrinol (1988) 2:95-100) incorporated herein byreference.

[0038] In addition to muteins of the alpha subunit which have alteredglycosylation patterns, a group of muteins with reduced or zero activityin signal transduction is also prepared. Experiments using chemicalderivatization in in vitro assays indicate that amino acids at positions88-92 (tyr-tyr-his-lys-ser) are necessary for the signal transductionactivity of the hormone. Accordingly, deletion or alteration of one ormore of these amino acids by site-directed mutagenesis results inanalogs which continue to bind to receptor but have reduced ornegligible activity. All four of the hormones sharing this alpha subunitcan thus be prepared as antagonists for the relevant hormone.

[0039] Both the wild type and mutant vectors can be used as a source ofhuman alpha subunit. Of particular importance are mutants of the alphasubunit in which the glycosylation site at Asn-52 is altered. Suchmutated sequences when ligated into expression systems and transfectedinto appropriate host cells result in produc- tion of proteins which,when combined with the appropriate beta subunit have antagonist activityfor the relevant hormone.

[0040] Construction of Expression Vectors for FSH Beta

[0041] The construction of expression vectors for FSH beta alone and forboth FSH beta and the human alpha subunit is shown in FIG. 3. The hostvectors, pM² and pM^(2/)α have been described previously. pM², asdescribed by Matzuk, M. M. et al., Proc Natl Acad Sci USA (1987)84:6354-6358, is a derivative of pSV2Neo and contains the ampicillinresistance gene (amp^(r)), the neomycin resistance gene (neo^(r)), andthe Harvey murine sarcoma virus long terminal repeat (LTR) promoter witha unique downstream BamHI site. The vector is diagrammed in FIG. 3.pM^(2/)α contains an alpha subunit mini gene downstream from a secondLTR. The construction of pM^(2/)α is described by Matzuk, M. M. et al.Mol Endocrinol (1988) 2:95-100 and the alpha subunit mini gene isdescribed by Matzuk, M. M. et al. J Cell Biol (1988) 106:1049-1058, bothincorporated herein by reference. This vector is also shown in FIG. 3.

[0042] For insertion of the inserted HindIII/BamHI fragment into eithervector, the 5′ HindIII site of FSH beta-containing pUC18 vector (PFSHbeta), was converted to a BglII site using oligonucleotide linkers, andthe modified pFSH beta vector digested with BglII and BamHI. Theresulting 3.7 kb BglII/BamHI fragment was inserted into the unique BamHIsite downstream of the LTR promoters in each vector, and orientation wasconfirmed by restriction analysis.

[0043] The foregoing constructions are, of course, merely illustrativeof expression vectors or systems which can be constructed for theproduction of FSH beta or its muteins alone or of the correspondingheterodimeric hormone. Alternate control sequences can be ligated to thecoding sequence of human FSH beta to effect expression in othereucaryotic cells which will provide suitable glycosylation. A variety ofcontrol sequences is known in the art, and methods to ligate the betaFSH coding sequence are of course also available. For example, suitableyeast promoters include promoters for synthesis of glycolytic enzymesincluding those for 3-phosphoglycerate kinase, or promoters from theenolase gene or the leu2 gene. Suitable mammalian promoters include theearly and late promoters from SV40, or other viral promoters such asthose derived from polyoma, adenovirus 2, bovine papilloma virus oravian sarcoma viruses. Suitable viral and mammalian enhancers can alsobe used. Expression in insect cells using a baculovirus promoter hasalso been reported. While less common, expression systems suitable forplant cells are also available.

[0044] A wide variety of expression vectors can be constructed utilizingvarious forms of the DNA encoding the desired amino acid sequence asshown in FIG. 2, or its alleles or modified (mutein) forms. The genomicDNA can be inserted directly into expression systems intended foreucaryotic host cells capable of processing introns. The nucleic acidsequences encoding the protein can be used directly from the genomicclone as described herein, or can be entirely or partially synthesizedusing standard solid phase oligonucleotide synthesis techniques asdescribed, for example, by Nambiar, K. P. et al. Science (1984) 223:1299or by Jaye, E. et al. J Biol Chem (1984) 259:6311. These techniques arenow commercially available. It is evident, of course, that not only thespecific nucleotide sequences shown in FIG. 2 can be employed, but alsonucleotide sequences employing codons which are degenerate with thoseshown.

[0045] In addition to expression vectors capable of reducing nativehuman FSH beta (i.e., that of the amino acid sequence shown in FIG. 2 orthe allelic variants thereof), the corresponding vectors capable ofexpressing genes encoding muteins of FSH beta are also constructed.

[0046] One important mutein encoding sequence is obtained by ligatingthe DNA sequence encoding the carboxy terminal extension peptide of CGbeta (CTP) to the 3′ end of the FSH beta encoding sequence. To theC-terminal Glu of FSH beta at position 111 is ligated the downstreamsequence of amino acid 112 to the carboxy terminus of CG beta, or avariant thereof. Preferred variants include those wherein the Ser atposition 112 of CG beta is replaced by Ala. The extended form isconveniently obtained by ligation of HindIII-digested FSH beta encodinginsert with the HindIII digest of CG beta cDNA. This religation resultsin the Ser → Ala substitution. The protein resulting from expression ofthis sequence when produced as the heterodimer FSH is expected to havethe biological activity of native FSH but a prolonged circulatinghalf-life. This expectation is made in view of the longer half-life ofCG as compared to LH, which is possibly ascribable to the presence of anumber of 0-linked glycosylation sites in the CTP as described by VanHall, E. Endocrinol (1971) 88:456. A major problem with FSH in clinicaluse is the relatively short circulating half-life of this protein (Wide,L. Acta Endocrinol (1986) 112:336).

[0047] Additional muteins of FSH beta are prepared by deleting oraltering the N-linked glycosylation sites represented by the Asn-Thrcombinations at positions 7/9 and 24/26 of the native sequence. Theprotein produced from expression of a system capable of expressing thegenes encoding these muteins is expected to show adequate receptorbinding with respect to the FSH beta receptor, and when heterodimerizedwith a suitable alpha subunit can be used as an antagonist for FSHactivity; or, when heterodimerized with a normal alpha subunit can beused as an FSH substitute.

[0048] Production of Human FSH with Glycosylation Defined by Host Choice

[0049] The expression systems constructed according to the precedingparagraph can be employed to produce FSH beta either alone or incombination with the human alpha subunit, so that the protein obtainedhas a glycosylation pattern which is internally consistent within thesample and which is characteristic of the recombinant host employed, andoptionally modified by changes in the glycosylation sites contained onthe amino acid sequence. Recombinant hosts suitable to the expressionsystem constructed must, of course, be employed.

[0050] With respect to the expression system illustrated in the aboveparagraph employing the Harvey murine sarcoma virus long terminalrepeat, suitable host cells are mammalian, in particular, host cellswhich are derived from rodents. A particularly preferred host cell,because of convenience and consistency in glycosylation pattern, is aChinese hamster ovary cell. For the illustrated expression systems,transfectants of CHO cells were obtained by transfection of CHO cellsaccording to the procedure of Matzuk, M. M. et al. (1987), supra, exceptthat the cells were maintained in alpha MEM containing 10% (v/v) bovinecalf serum, 100 U/ml penicillin and 100 ug/ml streptomycin as a growthmedium. Stable transfectants were selected from this growth mediumsupplemented with 250 ug/ml G418. Dimer-secreting clones (i.e., those 2/derived from transfection with pM^(2/)α into which FSH beta had beeninserted) were isolated by screening media and lysates with alpha andbeta antisera. Cultures transfected with pM² into which FSH beta wasinserted were screened by immunoprecipitation of lysates with FSH betaantisera.

[0051] Of course, the human alpha subunit can be expressed on a separatevector and, for example, pM^(2/)α or pM²CGα can be cotransfected withthe plasmid pM²FSHβ into CHO cells for synthesis of the dimeric hormone.

[0052] The expression systems described above for human FSH betainserted into pM² for expression of FSH beta alone or into pM^(2/)α forexpression in tandem with the alpha subunit were transfected into CHOcells and stable clones shown to express the beta subunit or dimer werecontinuously labeled with ³⁵S-cysteine for 6 hr. The proteins secretedinto the media and from cell lysates were immunoprecipitated withappropriate antisera and resolved on SDS-PAGE. The results are shown inFIG. 4 in comparison with the behavior of transformants expressing thegene for human CG beta.

[0053]FIG. 4a, which displays gels from 6 hr labeling, shows that in theabsence of the alpha subunit, FSH beta is retained in the lysate, while,as shown in FIG. 4b, when the alpha subunit is present, the dimer isformed and efficiently secreted into the medium. The results ofexperiments wherein the cells are pulse labeled with ³⁵S-cysteine for 20min and chased with unlabeled cysteine for up to 12 hr are shown in theremaining segments of FIG. 4. FIG. 4c shows the results for the betasubunit of CG where the lower molecular weight beta subunit in themedium is apparently due to the differences in the extent ofglycosylation at the 2 Asn-linked glycosylation sites on CG beta and isunique to this beta subunit. The half-life of CG beta from lysates andof appearance of CG beta in the medium are identical at about 2 hr andalmost all the secreted beta subunit can be recovered.

[0054]FIG. 4d shows that FSH beta alone is secreted much lessefficiently and as does CG beta, disappears from the cell lysates afterabout 5 hr; less than 20% is recovered in the medium after 12 hr.Similarly to the beta subunits of LH and TSH, FSH beta alone isinefficiently secreted and slowly degraded intracellularly. However,FIG. 4e shows that the presence of the alpha subunit stabilizes andenhances the secretion of the beta subunit for FSH. The half-life fordisappearance from the lysates was about 90 min, and 90% was recoveredin the medium after 12 hr. This behavior is similar to that shown forTSH above, but different from both CG and LH.

[0055] The transformants secreting dimer were tested for biologicalactivity and by chromatofocusing. Rat granulosa cells were treated withincreasing aliquots (0.01-1.0 ul/ml) of recombinant FSH-containingmedium in an in vitro assay for steroidogenesis as described by Jia, X.C., et al. J Clin Endocrinol Metab (1986) 621243-1249; Jia, X. C.Endocrinol (1986) 119:1570-1577. The results of this assay are shown inFIG. 5. These results show that maximum estrogen production was 10-foldhigher than basal values and similar to that induced by pituitary FSHstandard LER-907. Neither recombinant CG nor purified FSH beta alonestimulate estrogen production. The results show that the biologicallyactive FSH dimer is secreted at about 1.1±0.4 IU/10⁶ cells/24 hrcorresponding to a specific activity of 6600 IU/mg immunoreactive FSH.The cells thus secrete 500 ng FSH/10⁶ cells in 24 hr.

[0056] The medium from the transfected CHO cell cultures waschromatographed on a PBE-94 column with a pH gradient from 7.0-3.5 andthe FSH bioactivity in each fraction was determined based on the invitro assay described above. As a control, purified human FSH(NIADD-hFSH-1-3) was treated similarly. The results, shown in FIG. 6,indicate that both recombinant and isolated human FSH show one majorpeak of activity with pI values between 5.0-3.6 for recombinant FSH andbetween 5.2 and 3.6 for purified FSH. Pituitary FSH displayed aheterogeneous range of bioactive alkaline forms which was not seen inthe recombinant protein. The results from chromatofocusing clearlyindicate a uniform nonheterogeneous glycosylated form of the protein.

[0057] In a similar manner, vectors constructed with the wild type ormutant alpha subunit genes (see above) are constructed to obtain humanFSH with glycosylation patterns typical of those associated with therelevant host and/or with the altered alpha subunits. In addition, amutant CHO cell line deficient in N-acetyl glucosaminyl- transferase Iactivity, such as 15B, can be used to alter glycosylation in both thealpha and beta subunits of FSH or other heterodimeric hormones.

[0058] Influence of Glycosylation on Secretion of Human Alpha Subunit

[0059] The resultant alpha subunit expression systems constructed asdescribed in the paragraphs above were transfected into CHO cells usinga modification of the calcium phosphate method wherein cells wereselected for insertion of the plasmid DNA by growing in a culture mediumcontaining 0.25 mg/ml of G418. Resistant colonies were harvested elevendays after transfection and screened for expression of alpha subunit byimmunoprecipitation of media or lysates of the cells with theappropriate antiserum. The CHO cells were maintained in Ham's F12 mediumsupplemented with pen/strep and glutamine (2 mM) containing 5% v/v FCSat 37° C. in a humidified 5% CO₂ incubator; transfected clones weremaintained with the addition of 0.125 mg/ml G418.

[0060] For metabolic labeling, on day 0 the cells were placed into 12well dishes at 350,000 cells/well in 1 ml medium supplemented with 5%FCS. For continuous labeling experiments, the cells were washed twicewith cysteine-free medium supplemented with 5% dialyzed calf serum inplace of FCS and were labeled for 7-8 hr in 1 ml of cysteine-free mediumcontaining 5% dialyzed calf serum and 50 uCi/ml ³⁵S-cysteine (more than1,000 Ci/mmol). The cell lysates or media were then immunoprecipitatedand, if appropriate, treated with endoglycosidases as described byCorless, C. L. et al. J Cell Biol (1987) 104:1173-1181. Theimmunoprecipitates were resolved on 15% SDS polyacrylamide gels.

[0061] Using this analysis method, it was clear that the level ofglycosylation had an influence not only on the secretion of the alphasubunit, but also on its processing. The results are summarized in Table1: TABLE 1 Lysate Medium % Secreted αWT 23 kd 28 kd >95% αΔAsn1 orαΔThr1 20 kd 22 kd >95% αΔAsn2 or αΔThr2 20 kd 23.5 kd <20% αΔAsn(1 + 2)or αΔThr(1 + 2) 15 kd 15 kd ″50% αWT + tunicamycin 15 kd 15 kd >95%

[0062] As shown in Table 1, loss of the glycosylation at the position 78Asn glycosylation site resulted in a substantial decrease in theefficiency of secretion. Evidently additional glycosylation takes placeduring the secretion event as evident by the higher molecular weightfound in the medium. This was confirmed by treatment of the secretedforms with endoglycosidaseF which cleaves complex oligosaccharides inaddition to high mannose noncomplex and hybrid-type oligosaccharides.More than 95% of the secreted material is sensitive to endoglycosidaseF,but not to endoglycosidaseH which cleaves only high mannose noncomplexand hybrid-type oligosaccharides.

[0063] Pulse chase experiments performed as described in Matzuk, M. M.et al. J Cell Biol (1988) 106:1049-1059, incorporated herein byreference, shows that the somewhat lower levels of secreted αΔAsnl orαΔThr1 is due to clonal variation rather than differences in secretionor degradation rates. However, the mutants lacking glycosylation at thesecond (position 78) glycosylation site showed decreased secretion ratesand an increased degradation rate.

[0064] It is clear from these results that the glycosylation at position2 has a profound influence both on secretion rate and on theintracellular stability of the alpha subunit alone.

[0065] Influence of Alpha Subunit Glycosylation on Secretion of hCG

[0066] The influence of the glycosylation state of the alpha subunit onthe efficiency of assembly of the dimeric hormone hCG was also studiedin Matzuk, M. M. (supra).

[0067] In the clones wherein hCG beta is formed in excess of the alphasubunit, all of the wild type alpha subunit is mobilized into thedimeric form of the hormone for secretion. On the other hand, thosemutants which are missing oligosaccharide from position 52(glycosylation site 1) are deficient in the secretion of intact hCGdimer by virtue of altering the assembly and/or stability of the dimercomplex. However, loss of glycosylation at position 2 seems to have noeffect on assembly of the dimeric hormone. Removal of both glycosylationsites has an intermediate effect on assembly; the removal ofglycosylation from both sites seems to have a lesser effect on theability of the hormone to assemble than removal of the glycosylationfrom position 1 alone. In addition, the beta subunit of hCG stabilizesthe mutants at position 2 from degradation of the alpha subunit.

[0068] It is clear from the foregoing results that the glycosylationpattern of the alpha subunit determines both the ability of the alphasubunit itself to be secreted and its ability to dimerize with the betasubunit to form intact hormone.

[0069] As noted in the paragraph describing the production of alphasubunit muteins, certain designated amino acids in the carboxy-terminalportion of the alpha subunit are required for signal transductionactivity. Accordingly, inactivated alpha subunit is useful in theconstruction of antagonists by dimerization with the appropriate betasubunit of any of the hormones FSH, LH, CG and TSH.

[0070] In addition, it has been shown that FSH produced in CHO cellsdeficient in the glycosylation enzyme N-acetyl-glucosamine transferase-1(NAGT-) results in an Asn-linked (GLcNAc)₂ (mannose)₅ oligosaccharides.Production of FSH in CHO cells lacking CMP-sialic acid transport intothe Golgi apparatus (ST⁻) results in sialic acid deficient FSH.

[0071] However, it is clear that the influence of the glycoprotein alphasubunit on secretion of beta subunits of the four hormones in this groupdiffers depending on the nature of the beta subunit. Matzuk, M. M., etal. Molec Endocrinol (1988) 2:95-100, incorporated herein by reference,show that the presence of the alpha glycoprotein has a different effecton the secretion of human thyrotropin as opposed to human CG or LH. Ithas been shown that in the absence of the alpha subunit, CG beta isefficiently secreted, but TSH and LH beta subunits are slowly degradedintracellularly and less than 10% secreted into the medium. However, inthe presence of the alpha subunit, CG beta is also secreted efficientlyas the intact dimeric hormone while only 50% of LH beta appears in themedium as LH dimer. On the other hand, the alpha subunit efficientlycombines with TSH beta, since greater than 95% of this beta subunit wassecreted as the dimer. This demonstrates that the assembly of thedimeric hormone is dependent on the nature of both subunits.

[0072] As described in the paragraphs with regard to the construction ofexpression systems for FSH beta, mutein forms of FSH beta which aresuperior in circulating half-life can be produced by construction of amutein containing the CTP amino acid sequence at the carboxy terminus ofhuman CG beta. In addition, the N-linked glycosylation sites of the FSHbeta subunit can be deleted or altered without affecting receptorbinding activity.

[0073] Mutein forms of hCG are also included in the scope of theinvention. Various muteins of hCG containing deleted or altered N-linkedglycosylation sites are recombinantly produced by construction ofexpression systems analogous to those for FSH beta and for the alphasubunit from the suitably modified forms of the appropriate genes.Absence of any or all of the hCG N-linked oligosaccharides had only aminor effect on receptor affinity; with respect to the production ofcAMP and steroidogenesis, absence of N-linked oligosaccharides from CGbeta or from Asn-78 of the alpha subunit had no effect. However, theoligosaccharide at asparagine-52 of alpha was critical for cAMP andsteroid production. In addition, its absence unmasked differences in thetwo N-linked oligosaccharides present in CG beta and inhibited in vitrobiological activity.

1. A recombinant expression system capable, when transformed into arecombinant host cell, of expressing the gene encoding human folliclestimulating hormone (FSH) beta subunit or mutein thereof.
 2. Theexpression system of claim 1 wherein said expression is effected bycontrol sequences which comprise a promoter functional in mammaliancells.
 3. The expression system of claim 2 wherein said promoter isderived from Harvey murine sarcoma virus.
 4. A recombinant host celltransfected with the expression system of claim 1 .
 5. A method toproduce human FSH beta which comprises culturing the cells of claim 4under conditions wherein the gene encoding human FSH beta is expressed,and recovering human FSH beta from the cell culture.
 6. The method ofclaim 5 wherein the recombinant host cells are Chinese hamster ovary(CHO) cells.
 7. The method of claim 6 wherein the CHO cells are mutantsdeficient in N-acetyl glucosaminyltransferase I activity.
 8. The methodof claim 7 wherein the mutant CHO cells are CHO-15B mutants.
 9. HumanFSH beta or mutein thereof having a glycosylation pattern characteristicof human FSH beta or said mutein produced by the method of claim 6 . 10.Human FSH beta or mutein thereof having a glycosylation patterncharacteristic of human FSH beta or said mutein produced by the methodof claim 7 .
 11. Human FSH beta mutein which consists essentially of theamino acid sequence of native FSH beta extended at the C terminus by theCTP sequence downstream of amino acid 111 of human CG beta.
 12. Arecombinant expression system capable, when transformed into arecombinant host cell of expressing a gene encoding a mutein of thehuman glycoprotein alpha subunit.
 13. The expression system of claim 12wherein said gene encodes a human alpha subunit mutein lacking aglycosylation site at position 52 or 78 or both.
 14. The expressionsystem of claim 12 wherein said gene encodes a human alpha subunitmutein lacking one or more of amino acids 88-92 of the native subunit.15. The expression system of claim 12 wherein said expression iseffected by control sequences which comprise a promoter functional inmammalian cells.
 16. A recombinant host cell transfected with theexpression system of claim 12 .
 17. A method to produce human alphasubunit which comprises culturing the cells of claim 16 under conditionswherein the gene encoding human alpha subunit mutein is expressed, andrecovering human alpha subunit mutein from the cell culture.
 18. Themethod of claim 17 wherein the alpha subunit mutein lacks aglycosylation site at position 52 or 78 or both.
 19. The method of claim17 wherein the alpha subunit mutein lacks one or more of amino acids88-92 of the native subunit.
 20. The method of claim 17 whereinrecombinant host cells are Chinese hamster ovary (CHO) cell mutantsdeficient in N-acetyl glucosaminyltransferase I activity.
 21. The cellsof claim 20 wherein the mutant CHO cells are CHO-15B mutants.
 22. Humanalpha subunit having a glycosylation pattern characteristic of humanalpha subunit produced by the cells of claim 20 .
 23. A method toproduce human alpha subunit which comprises culturing the cells of claim21 under conditions wherein the gene encoding human alpha subunit isexpressed, and recovering human alpha subunit from the cell culture. 24.Human alpha subunit having a glycosylation pattern characteristic ofhuman alpha subunit produced by the method of claim 23 .
 25. A humanalpha subunit mutein.
 26. The mutein of claim 25 which does not confersubstantial signal transducing activity when heterodimerized but bindsto receptor when heterodimerized.
 27. The mutein of claim 26 whichcontains at least one altered amino acid or at least one deletion inpositions 88-92.
 28. The host cell of claim 27 which is furthertransfected with a second recombinant expression system capable ofeffecting the production of the beta subunit of human FSH, LH, TSH orCG.
 29. The host cell of claim 27 wherein the second recombinantexpression system is capable of effecting production of FSH beta or amutein thereof from a gene encoding FSH beta or said mutein.
 30. Amethod to produce human FSH which comprises culturing the cells of claim29 under conditions wherein the genes encoding human FSH beta or saidmutein and human alpha subunit are expressed and recovering human FSHfrom the cell culture.
 31. The host cell of claim 27 wherein the secondrecombinant expression system is capable of effecting production of LHbeta from a gene encoding LH beta.
 32. A method to produce LH whichcomprises culturing the cells of claim 31 under conditions wherein thegenes encoding human LH beta and human alpha subunit are expressed andrecovering human LH from the cell culture.
 33. The host cell of claim 27wherein the second recombinant expression system is capable of effectingproduction of TSH beta from a gene encoding TSH beta.
 34. A method toproduce TSH which comprises culturing the cells of claim 33 underconditions wherein the genes encoding human TSH beta and human alphasubunit are expressed and recovering human TSH from the cell culture.35. The host cell of claim 27 wherein the second recombinant expressionsystem is capable of effecting production of CG beta from a geneencoding CG beta.
 36. A method to produce CG which comprises culturingthe cells of claim 35 under conditions wherein the genes encoding humanCG beta and human alpha subunit are expressed and recovering human CGfrom the cell culture.
 37. Human FSH having a glycosylation patterncharacteristic of human FSH prepared by the method of claim 30 . 38.Human LH having a glycosylation pattern characteristic of human LHprepared by the method of claim 32 .
 39. Human TSH having aglycosylation pattern characteristic of human TSH prepared by the methodof claim 34 .
 40. Human CG having a glycosylation pattern characteristicof human CG prepared by the method of claim 36 .
 41. A pharmaceuticalcomposition useful in treating reproductive disorders in human subjectswhich comprises the glycoprotein of claim 37 in admixture with asuitable pharmaceutically acceptable excipient.
 42. A method to regulatereproductive metabolism in humans which comprises administering to asubject in need of such regulation an effective amount of theglycoprotein of claim 37 or a pharmaceutical composition thereof.
 43. Apharmaceutical composition useful in treating reproductive disorders inhuman subjects which comprises the glycoprotein of claim 38 in admixturewith a suitable pharmaceutically acceptable excipient.
 44. A method toregulate reproductive metabolism in humans which comprises administeringto a subject in need of such regulation an effective amount of theglycoprotein of claim 38 or a pharmaceutical composition thereof.
 45. Apharmaceutical composition useful in treating reproductive disorders inhuman subjects which comprises the glycoprotein of claim 40 in admixturewith a suitable pharmaceutically acceptable excipient.
 46. A method toregulate reproductive metabolism in humans which comprises administeringto a subject in need of such regulation an effective amount of theglycoprotein of claim 40 or a pharmaceutical composition thereof.